Bottom Line:
In the present study, we demonstrated that astrocyte elevated gene-1(AEG-1) ectopic overexpression promoted EMT, which resulted from the down-regulation of E-cadherin and up-regulation of Vimentin in lung cancer cell lines and clinical lung cancer specimens.Using an orthotopic xenograft-mouse model, we also observed that AEG-1 overexpression in human carcinoma cells led to the development of multiple lymph node metastases and elevated mesenchymal markers such as Vimentin, which is a characteristic of cells in EMT.Furthermore, AEG-1 functioned as a critical protein in the regulation of EMT by directly targeting multiple positive regulators of the Wnt/β-catenin signaling cascade, including GSK-3β and CKIδ.

Background: Non-small cell lung cancer (NSCLC) is a highly metastatic cancer with limited therapeutic options, so development of novel therapies that target NSCLC is needed. During the early stage of metastasis, the cancer cells undergo an epithelial-mesenchymal transition (EMT), a phase in which Wnt/β-catenin signaling is known to be involved. Simultaneously, AEG-1 has been demonstrated to activate Wnt-mediated signaling in some malignant tumors.

Methods: Human NSCLC cell lines and xenograft of NSCLC cells in nude mice were used to investigate the effects of AEG-1 on EMT. EMT or Wnt/β-catenin pathway-related proteins were characterized by western blot, immunofluorescence and immunohistochemistry.

Results: In the present study, we demonstrated that astrocyte elevated gene-1(AEG-1) ectopic overexpression promoted EMT, which resulted from the down-regulation of E-cadherin and up-regulation of Vimentin in lung cancer cell lines and clinical lung cancer specimens. Using an orthotopic xenograft-mouse model, we also observed that AEG-1 overexpression in human carcinoma cells led to the development of multiple lymph node metastases and elevated mesenchymal markers such as Vimentin, which is a characteristic of cells in EMT. Furthermore, AEG-1 functioned as a critical protein in the regulation of EMT by directly targeting multiple positive regulators of the Wnt/β-catenin signaling cascade, including GSK-3β and CKIδ. Notably, overexpression of AEG-1 in metastatic cancer tissues was closely associated with poor survival of NSCLC patients.

Conclusions: These results reveal the critical role of AEG-1 in EMT and suggest that AEG-1 may be a prognostic biomarker and its targeted inhibition may be utilized as a novel therapy for NSCLC.

Fig6: AEG-1 expression levels were closely correlated with risk of lymph node metastasis in primary cancer lung cancer. (A) H&E staining was used to identify lung cancer patients with or without lymph node metastasis (magnification × 200). (B) AEG-1 regulates EMT in the different stages of lung cancer, including lung cancer patients with or without metastasis, as determined by positron emission tomography/computed tomography. (C) AEG-1 over-expression closely correlates with changes in the EMT marker in clinical specimens from lung cancer patients. Expression levels of AEG-1, E-cadherin, Vimentin, p-GSK-3β (Ser-9), GSK-3β and β-catenin in normal (n = 3) and lung cancer (n = 3) tissues were determined by Western blotting. Densitometry was used to determine relative protein levels, and all proteins were normalized to the levels of GAPDH.

Mentions:
To further understand the clinical relevance of the above findings, we examined the relationship between AEG-1 expression and EMT markers in lung cancer patients. Patients from different clinical stages were first divided into two groups according to H&E staining (Figure 6A) and positron emission tomography/computed tomography (PET/CT) (Figure 6B): the primary site of cancer with metastasis and the primary site of non-metastasizing cancer, respectively. Based on the TNM (Tumor node metastasis) staging system, we selected six patients from stage I and stage IV. As shown in Figure 6C, the expression levels of AEG-1 were significantly elevated in patients with distant metastasis, compared to that in primary tumors without detectable distant metastasis. Furthermore, up-regulation of AEG-1, Vimentin, p-GSK-β, and β-catenin levels, as well as suppression of E-cadherin, were clearly observed in tissues from patients with distant metastasis (Figure 6C). In all six examined samples, there was a significantly positive correlation between the levels of AEG-1 and Vimentin and an inverse correlation between the levels of AEG-1 and E-cadherin. These data indicate that AEG-1 plays a pivotal role in lung cancer EMT and metastasis in vivo, which is consistent with our in vitro data from various cancer cell lines.Figure 6

Fig6: AEG-1 expression levels were closely correlated with risk of lymph node metastasis in primary cancer lung cancer. (A) H&E staining was used to identify lung cancer patients with or without lymph node metastasis (magnification × 200). (B) AEG-1 regulates EMT in the different stages of lung cancer, including lung cancer patients with or without metastasis, as determined by positron emission tomography/computed tomography. (C) AEG-1 over-expression closely correlates with changes in the EMT marker in clinical specimens from lung cancer patients. Expression levels of AEG-1, E-cadherin, Vimentin, p-GSK-3β (Ser-9), GSK-3β and β-catenin in normal (n = 3) and lung cancer (n = 3) tissues were determined by Western blotting. Densitometry was used to determine relative protein levels, and all proteins were normalized to the levels of GAPDH.

Mentions:
To further understand the clinical relevance of the above findings, we examined the relationship between AEG-1 expression and EMT markers in lung cancer patients. Patients from different clinical stages were first divided into two groups according to H&E staining (Figure 6A) and positron emission tomography/computed tomography (PET/CT) (Figure 6B): the primary site of cancer with metastasis and the primary site of non-metastasizing cancer, respectively. Based on the TNM (Tumor node metastasis) staging system, we selected six patients from stage I and stage IV. As shown in Figure 6C, the expression levels of AEG-1 were significantly elevated in patients with distant metastasis, compared to that in primary tumors without detectable distant metastasis. Furthermore, up-regulation of AEG-1, Vimentin, p-GSK-β, and β-catenin levels, as well as suppression of E-cadherin, were clearly observed in tissues from patients with distant metastasis (Figure 6C). In all six examined samples, there was a significantly positive correlation between the levels of AEG-1 and Vimentin and an inverse correlation between the levels of AEG-1 and E-cadherin. These data indicate that AEG-1 plays a pivotal role in lung cancer EMT and metastasis in vivo, which is consistent with our in vitro data from various cancer cell lines.Figure 6

Bottom Line:
In the present study, we demonstrated that astrocyte elevated gene-1(AEG-1) ectopic overexpression promoted EMT, which resulted from the down-regulation of E-cadherin and up-regulation of Vimentin in lung cancer cell lines and clinical lung cancer specimens.Using an orthotopic xenograft-mouse model, we also observed that AEG-1 overexpression in human carcinoma cells led to the development of multiple lymph node metastases and elevated mesenchymal markers such as Vimentin, which is a characteristic of cells in EMT.Furthermore, AEG-1 functioned as a critical protein in the regulation of EMT by directly targeting multiple positive regulators of the Wnt/β-catenin signaling cascade, including GSK-3β and CKIδ.

Background: Non-small cell lung cancer (NSCLC) is a highly metastatic cancer with limited therapeutic options, so development of novel therapies that target NSCLC is needed. During the early stage of metastasis, the cancer cells undergo an epithelial-mesenchymal transition (EMT), a phase in which Wnt/β-catenin signaling is known to be involved. Simultaneously, AEG-1 has been demonstrated to activate Wnt-mediated signaling in some malignant tumors.

Methods: Human NSCLC cell lines and xenograft of NSCLC cells in nude mice were used to investigate the effects of AEG-1 on EMT. EMT or Wnt/β-catenin pathway-related proteins were characterized by western blot, immunofluorescence and immunohistochemistry.

Results: In the present study, we demonstrated that astrocyte elevated gene-1(AEG-1) ectopic overexpression promoted EMT, which resulted from the down-regulation of E-cadherin and up-regulation of Vimentin in lung cancer cell lines and clinical lung cancer specimens. Using an orthotopic xenograft-mouse model, we also observed that AEG-1 overexpression in human carcinoma cells led to the development of multiple lymph node metastases and elevated mesenchymal markers such as Vimentin, which is a characteristic of cells in EMT. Furthermore, AEG-1 functioned as a critical protein in the regulation of EMT by directly targeting multiple positive regulators of the Wnt/β-catenin signaling cascade, including GSK-3β and CKIδ. Notably, overexpression of AEG-1 in metastatic cancer tissues was closely associated with poor survival of NSCLC patients.

Conclusions: These results reveal the critical role of AEG-1 in EMT and suggest that AEG-1 may be a prognostic biomarker and its targeted inhibition may be utilized as a novel therapy for NSCLC.